Vitis 34 (1), 37-39 (1995)
ItAPD markers in wild, and cultivated Vitis vinifera
S u m m a r y : Some ~tis vinifera cultivars and V. vinifera ssp. silvestris individuals have been subjected to the RAPD analysis in order to estimate the genetic diversity existing within this germplasm. 44 decamer primers of arbitrary sequence have been used for PCR and reproducible band profiles have been obtained. The distribution of the individualized polymorphic DNA markers has not turned out to be different in a remarkable way between cultivated and wild grapevines but this RAPD approach provides for some characteristics useful to analyze genetic relationships even within the ~tis vinifera species.
K e y w o r d s : genetic diversity, PCR, Vitis vinifera ssp. silvestris.
Introduction
Some years ago a research on the diffusion and char-acterization of the Italian populations of ~tis vinifera ssp. silvestris was set up. Among one of its aims there was also the analysis of the relationships between wild and culti-vated grapevines(SCIENZA 1983).
The individualized and collected germplasm of V. viniferassp . silvestris has been described by means of the usual ampelographic method and through ampelometric analyses (ANZANI et al. 1990, FAILLA et al. 1992). Some
genotypes have also been characterized according to the electrophoresic patterns of the seed storage proteins
(SCIENZA et al. 1990) and pollen wall proteins (TEDESCO
et al 1990) as well as according to the anthocyanin pro-file of grape skins(MATTIVIet al. 1990, and 1993,VALENTI
et al. 1993).
Since the molecular analysis methods based upon PCR (Polymerase Chain Reaction), recently applied also to the DNA of grapevine, can give a practically unlimited series of primary characters(GRANDOet al. 1994), we suggest in
this paper the attempt of using RAPD markers (Random Amplified Polymorphic DNA) (WELSH and MCCLELLAND
1990,WILLIAMSet al. 1990) as an approach to the study of
the genetic diversity of wild and cultivated grapevines.
Materials and methods
G r a p e m a t e r i a l : All plant material was obtained from the germplasm collection maintained at our Institute. Young fully expanded leaves were harvested, im mediately frozen in liquid nitrogen and stored at -80 °C until DNA extraction. The genotypes used for this analy-sis are listed in the Table.
D N A i s o l a t i o n : DNA was extracted and purified according to the hexadecyltrimethylammonium bromide (CTAB) method OfDOYLEandDOYLE (1987) for
fresh tissue. To estimate DNA concentration, samples of by
M.S. GRANDO, L. DE MICHELI, L. BIASETTOand A. SCIENZA
Istituto Agrario, San Michele alfAdige, Italia
grape DNA were compared with dilutions of known con-centration of ,DNA after agarose gel electrophoresis and ethidium bromide staining.
Amplification reaction conditions 44 random decamer oligonucleotides (from sets A, E and U, Operon Technologies Inc. Alameda CA, USA), were used as single primers for the amplification of RAPD se-quences. The polymerase chain reaction (PCR) was per-formed in a volume of 25 pl containing 5 ftl of the DNA extract (approximately 15 ng), 10 mM Tris-HCl pH 9, 50 mM KCI, 2 mM MgCJ~, 0.1 mM each dATP, dCTP, dGTP and dTTP (Boehringer), 0.28 pM primer and 0.3 U Taq DNA polymerase (Super Taq P.H. Stehelin & Cie AG, Basel, Switzerland) . Amplification was performed in a Perkin Elmer Cetus Gene Amp PCR System 9600 pro-grammed as follows(KOLLERet al. 1993): 2 cycles of 30 s
at 94 °C, 30 s at 36 °C, 120 s at 72 °C; 20 cycles of 20 s at 94 °C, 15 s at 36 °C, 15 s at 45 °C, 90 s at 72 °C; 19 cycles of 20 s at 94 °C (increased 1 s/cycle), 15 s at 36 °C, 15 s at 45 °C, 120 s at 72 °C (increased 3 s/cycle) followed by
10 min at 72°C.
List of cultivars and individuals of ~tis vinifera used for the RAPD analysis
Correspondence to: Dr. M.S. GRANDO, Istituto Agrario, I-38010 San Michele alfAdige, Italy
Table
CULTIVATED WILD
CODE ORIGIN
Armenia va03 Yeni Katla Germany Bombino n . va12 K1-HOerdt Germany Chardonnay va13 Sinni 2 Italy Chenin b . ve16 Moille Switzerland
Furmint va17 Sinni Italy
Garganega va18 Veano 6 Italy
H~rslevel~3 vs24 Sinni T Italy Kadarka va25 Casale Giglio Italy Lambruaco f .f . va26 Hagnoli Italy Muscat of Alexandria vs27 Sinni P Italy
Pinot n . vs29 Nigra Germany
Riesling r . va31 Caucasica URSS Rkatsiteli vs33 Sinni 5 Italy Sangiovese vs37 Cumone 1 Italy Sauvignon b . vs38 Sinni U Italy Semillon va42 Fenelle Switzerland
Syrah va43 Nettuno Italy
Tocai friulano va56 Martignano Italy Trebbiano toscano
38
After the cycling was completed, 12 ltl of the RAPD products were separated by electrophoresis in 1.5 % agarose gels run in 0.5 x TBE (0.045 M Tris-borate and 1.0 mM EDTA pH 8), visualized with ethidium bromide and pho-tographed under UV with Polaroid 667 films.
D a t a a n a l y s i s : Each amplified band was named by the primer used and its size in bp. RAPD pro-files were scored visually. Data were recorded as the pres ence or absence of an amplification fragment of a given length. Only those major fragments whose presence was unambiguous were retained.
Genetic similarity between each pair of accessions and the dendrogramtree were calculated using NTSYS-pc pack-age 1 .8 developed by Rohlf (Exeter Software, New York, USA).
Results and discussion
The DNA amplification conditions we used, have pro-duced DNA fragments of discrete size with all tested prim-ers. Yet, the electrophoresic profiles have turned out to be quite complex and often show not very consistent poly-morphic bands. Anyway, a good reproducibility of the sys-tem has been noted, both concerning the number, and the intensity of the amplification products (Fig. 1) . The 2 groups of primers belonging to sets A and E have shown a clear difference in their capacity of generating polymor-phic DNA fragments. Among the 20 primers of set E no RAPD markers with a clear position in gel have been de-termined, whereas set A has given most of the polymor-phic products used for statistical analysis. From set U prim-ers 1, 2, 10 and 14 have been tested. The latter has given 2 poymorphic bands.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 l7 18 19 20 21 22
Fig. 1 : RAPD profiles generated by primer U14 (5'TGGGT000TC3') . For some accessions the DNA products oftwo independent reactions are shown. 1 Tocai f., 2 Sangiovese, 3 Sauvignon b.> 4 Chenin b., 5 Semillon, 6 Harslevelu, 7 Syrah, 8 Armenia, 9 Rkatsiteli , 10-18 vs 27, 11-19 vs 31, 12-20 vs 56, 13-21 vs 12, 14-22 vs 24, 15 vs 29, 16 vs 33, 17 vs 16> M
Mo-lecular size DNA Ladder (Gibco BRL).
The comparison between genotypes has thus been based upon the distribution of the following RAPD mark ers: Al9es° A18~, A783o> A8,ozo+ A9szo~ A94so+ AlO,s~o> U149so and U14s$s (Figs. 1, 2, 3). The dendrogram originated from this first comparative analysis of RAPD profiles (Fig. 4) has not pointed out remarkable differences between culti-vated and wild grapevines. Nevertheless, it can be noted that some vines ascribed to the occidentalis NEGR. group
are clearly separated from the other genotypes whereas
M.S. GRANDO, L. DE MICHELI, L. BIASETTOand A. SCIENZA
the RAPD profiles of V.vinifera ssp. silvestris accessons are very often more similar or even identical to those of the cultivars ascribed to theproles pontica NECR.group.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Fig . 2: RAPD profiles generated by primer A01 (5'AG000CTTC3') . 1-9 and M: see Fig. 1 ; 10 vs 56, 11 vs 12,
12 vs 24, 13 vs 29, 14 vs 33, 15 vs 16.
MI 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18M
Fig. 3 : RAPD profiles generated by primer A07 (5'GAAAC000TG 3'). 1-9 and M: see Fig. 1 ; 10 vs 27, 11 vs 31, 12 vs 56, 13 vs 12, 14 vs 13, 15 vs 24, 16 vs 29, 17 vs 33, 18 vs16. 0.25 0.50 0.75 1 .00 Conclusions YSYI vs31 -- vs3 ~.~ Kadarca Muscat A vs29 Furnint vsf2 Yeiz vs16 _ Rkatsiteli HSrlevelu vs17 vs26 l.anbrus[o_ff vs33 rs37 --'~ vs3B ys25 Pinat n --- vs56 ---- vs2f --- Rr nenia --- vs43 ,.~{ io[ai_F rrebbiano ----°- Syr ah --- vs13 ^-- Bonbino-n -- vsi S --- 5angi ovese --- 6arganega ~~- Chardonnay Riesling_ ,~____~~ 6auvignon Chenin --- Seni l L on
Fig. 4: Phylogram tree based on RAPDs data showing the rela-tionships among Vitis vinifera wild and cultivated genotypes.
Through this first screening of oligo-nucleotide decamers of arbitrary sequence for a RAPD analysis of the germplasm of cultivated and wild V. vinifera, some
polymorphisms of the DNA amplification products have been selected. Although for some DNA markers a greater homogeneity in the accessions of the V. vinifera ssp. silvestris group has been noted, no remarkable differences between cultivated and wild vines have been found.
Yet, the different capacity of generating polymorphic markers as shown by the different sets of primers used and the good quality of the analysis procedure used, encour age us to go on using the screening system in order to base the comparison between accessions on larger portions of V. vinifera genome.
Acknowledgement
Researches supported by the Italian Ministry of Agriculture in the framework of the project "Resistenze genetiche delle piante agrarie agli stress biotici ed abiotici".
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Received September 16, 1994
